FLOW CHAMBER FOR MICROSCOPY

Abstract

A flow chamber for live-cell microscopic observation. The flow chamber includes a top portion having at least two perfusion tubes and a central aperture throughout. A pressure distribution/perfusion ring is adjacent to the top portion. An upper gasket engages and seals the pressure distribution/perfusion ring and the microaqueduct slide. The microaqueduct slide is placed between the upper gasket and the lower gasket. A cover slip is arranged intermediate the lower gasket, and a base. A flow chamber is formed between the cover slip, the microaqueduct slide, and the lower gasket. The flow chamber is in fluid communication with the at least two perfusion tubes in the top portion. The microaqueduct slide has perfusion grooves and micro-channels are formed through the microaqueduct slide and through the upper and lower gaskets. The base has a raised rim portion to receive the pressure distribution/perfusion ring in a non-rotational fit.

Claims

1. A flow chamber for studying live-cell microscopic observation comprising: at least two perfusion tubes and a central aperture therethrough, the central aperture extending through a fluid optical cavity to provide illumination and facilitate observation; a pressure distribution/perfusion ring, a coverslip, an upper gasket, a lower gasket and a microaqueduct slide disposed between the upper gasket and the lower gasket; the upper gasket operatively engaging and sealing the pressure distribution/perfusion ring; the coverslip and the micro aqueduct slide separated by the upper gasket having a specific central aperture and defining an optical cavity therebetween; the pressure distribution/perfusion ring disposed adjacent to a top portion; and having a predetermined geometry to facilitate a fluidic transfer through the optical cavity; a cover slip positioned adjacent the lower gasket opposite the microaqueduct slide and operatively disposed intermediate microaqueduct slide the lower gasket and a receiver platform; a fluid optical cavity in fluid communication with the at least two perfusion tubes in the top portion; wherein the microaqueduct slide further comprises a plurality of perfusion grooves formed in the microaqueduct slide, and a plurality of micro-channels formed through the microaqueduct slide bordered by the lower gasket unit; and wherein the base having a raised rim portion defining a peripheral geometric recess complementary to the geometric shape of the pressure distribution/perfusion ring; the recess arranged to receive the pressure distribution/perfusion ring in a non-rotational fit.

2. The flow chamber of claim 1, wherein the pressure distribution/perfusion ring is configured to apply a uniform pressure to the upper gasket, the microaqueduct slide, the lower gasket and the coverslip.

3. The flow chamber of claim 2, wherein the pressure distribution/perfusion ring, the upper gasket, the microaqueduct slide, the lower gasket and the coverslip are arranged compressed uniformly into a base.

4. The flow chamber of claim 1, wherein the upper gasket operatively engages and seals the pressure distribution/perfusion ring to the microaqueduct slide.

5. The flow chamber of claim 1, wherein the microaqueduct slide is disposed between the upper gasket and the lower gasket.

6. The flow chamber of claim 1, wherein the cover slip is disposed adjacent the lower gasket and opposite the microaqueduct slide.

7. The flow chamber of claim 1, wherein the cover slip is operatively disposed intermediate the microaqueduct slide, the lower gasket and a base.

8. The flow chamber of claim 1, wherein the flow chamber is formed between the cover slip, and microaqueduct slide, by the lower gasket.

9. The flow chamber of claim 1, wherein the fluid optical cavity is in fluid communication with the at least two perfusion tubes in the pressure distribution/perfusion ring.

10. The flow chamber of claim 1, wherein the microaqueduct slide comprises a plurality of perfusion grooves, and micro-channels formed through the microaqueduct slide and bordered by the lower gasket unit.

11. The flow chamber of claim 1, wherein the base comprises a raised rim portion defining a peripheral geometric recess complementary to the geometric shape of the pressure distribution/perfusion ring, gaskets, and microaqueduct slide.

12. The flow chamber of claim 11, wherein the recess is arranged to receive the distribution/perfusion ring in a non-rotational friction fit.

13. The flow chamber of claim 1, wherein the recess is arranged to receive the distribution/perfusion ring in a non-rotational friction fit.

14. The flow channel of claim 1 wherein the base is attachable to a microscope and supported by a stage adapter.

15. A perfusion ring assembly for a flow chamber comprising: a pressure distribution/perfusion ring, a coverslip, an upper gasket, a lower gasket and a microaqueduct slide disposed between the upper gasket and the lower gasket; the upper gasket operatively engaging and sealing the pressure distribution/perfusion ring; the coverslip and the micro aqueduct slide separated by the upper gasket having a specific central aperture and defining an optical cavity therebetween; the pressure distribution/perfusion ring disposed adjacent to the top portion; and having a predetermined geometry to facilitate a fluidic transfer through the optical cavity; a cover slip positioned adjacent the lower gasket opposite the microaqueduct slide and operatively disposed intermediate the microaqueduct slide and a receiver platform; a fluid optical cavity formed between the cover slip, the microaqueduct slide, and the lower gasket; wherein the fluid optical cavity is in fluid communication with the at least two perfusion tubes in the top portion.

16. The perfusion ring assembly of claim 15, wherein the microaqueduct slide further comprises a plurality of perfusion grooves formed in the microaqueduct slide, and a plurality of micro-channels formed through the microaqueduct slide.

17. The perfusion ring assembly of claim 15, wherein the lower gasket unit and the upper seal unit further comprise a plurality of perfusion grooves.

18. The perfusion ring assembly of claim 15, wherein the base comprises a raised rim portion defining a peripheral geometric recess complementary to the geometric shape of the pressure distribution/perfusion ring; the recess arranged to receive the pressure distribution/perfusion ring in a non-rotational friction fit.

19. A perfusion ring assembly for a flow chamber comprising: a pressure distribution/perfusion ring, a coverslip, an upper gasket, a lower gasket and a microaqueduct slide disposed between the upper gasket and the lower gasket; the upper gasket operatively engaging and sealing the pressure distribution/perfusion ring; the coverslip and the micro aqueduct slide separated by the upper gasket having a specific central aperture and defining an optical cavity therebetween; the pressure distribution/perfusion ring disposed adjacent to the top portion; and having a predetermined geometry to facilitate a fluidic transfer through the optical cavity; a cover slip positioned adjacent the lower gasket opposite the microaqueduct slide and operatively disposed intermediate the microaqueduct slide and a receiver platform; a fluid optical cavity formed between the cover slip, the microaqueduct slide, and the lower gasket; wherein the fluid optical cavity is in fluid communication with the at least two perfusion tubes in the top portion; and a microscope specimen warmer; wherein the microscope specimen warmer comprises: a) a plate structure having minimal thermal conductivity and a minimal expansion coefficient; b) a support surface on the plate structure; and c) a specimen resting surface that is supported by the support surface on the plate structure so that the support surface on the plate structure is at or near the same horizontal plane as a specimen within the specimen resting surface so that there is no detectable z-axis shift of the specimen when the specimen is heated.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The application will become more fully understood from the following detailed description, taken in conjunction with the accompanying figures, wherein like reference numerals refer to like elements, in which:

[0025] FIG. 1 is an exploded perspective view of the flow chamber for live-cell microscopic observation.

[0026] FIG. 2 is a perspective view of the novel specimen heating and retaining assembly of the present invention shown in use on the stage of a standard inverted microscope. A specimen dish and a heating element that does not form a part of the present invention are illustrated in phantom.

[0027] FIG. 3 is a perspective cross-sectional view of the novel specimen heating and retaining assembly of the present invention along the line II-II of FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Before turning to the figures which illustrate the exemplary embodiments in detail, it should be understood that the application is not limited to the details or methodology set forth in the following description or illustrated in the figures. It should also be understood that the phraseology and terminology employed herein is for the purpose of description only and should not be regarded as limiting.

[0029] Referring to FIG. 1, a flow chamber, generally designated as 10, is shown in an exploded perspective view. Flow chamber 10 includes a top portion 12 having a central aperture for illumination and observation of specimens plated on or contained within the optical cavity defined between the micro aqueduct slide 18 and the coverslip 32. Two or more perfusion tubes 14 extend from the sides of a pressure distribution/perfusion ring 22. The perfusion tubes 14 in fluid communication with laminar flow ports, or perfusion grooves 20 on microaqueduct slide 18. Pressure distribution/perfusion ring 22 is located under top portion 12 having a rotational torque indicator. The pressure distribution/perfusion ring 22 defines a circular aperture and having squared peripheral sides for alignment with a receiver platform, or base 24. Pressure distribution/perfusion ring 22 applies uniform pressure to components 26, 18, 28, and 32 against platform 24.

[0030] An upper gasket 26 is disposed between pressure distribution/perfusion ring 22 and microaqueduct slide 18 to provide a liquid-tight sealed periphery with pressure distribution/perfusion ring 22. Upper gasket 26 comprises a generally circular aperture with squared sided geometry and made of, thin resilient member preferably fabricated from an inert material such as rubber, or silastic sheeting. Upper gasket 26 is dimensioned to be received in a lower side of pressure distribution/perfusion ring 22. In addition, upper gasket 26 has a central aperture with approximately the same radius as aperture 16. Microaqueduct slide 18 rests on lower gasket 28. Lower gasket 28 includes a viewing aperture. The aperture 30 may take any geometric shape, e.g., rectangular, square, round, oval, or other desired shape. Preferably lower gasket 28 may have a thickness ranging from 0.01 millimeter (mm) to 2.0 mm to allow the volume and flow characteristics to be customized for the flow chamber 10. Similarly, lower gasket 28 is a thin, resilient member preferably fabricated from an inert material such as rubber, or silastic sheeting.

[0031] Stage adapter 36 comprises a generally rectangular rigid support plate member, typically machined from aluminum in a geometry to be compatible with the microscope stage. Central aperture 37 may be defined by inwardly beveled side walls that are dimensioned to engage beveled edges of platform 24. Stage adapter 36 may be further provided with securing means adapted to operatively engage platform 24.

[0032] Flow chamber 10 provides a closed system, live-cell micro-observation chamber 10. Flow chamber 10 provides uniform temperature control and user definable perfusion capability that is fully compatible with all modes of microscopy. It is also the only chamber to combine high-volume laminar flow perfusion rates with Koehler illumination compatibility as well as the capability of precise temperature control without the need for external peripheral temperature control. This is accomplished by both internal heating of the platform 24 and electronic control of the Microaqueduct slide 18. High-volume laminar flow perfusion rates are the result of the development of micro-aqueduct perfusion. Perfusion introduces media into a fluid optical cavity through the grooves in the Microaqueduct slide. Therefore the separation of optical surfaces and flow geometry is determined by the thickness and internal geometry of a single gasket without the need of an additional component to facilitate flow. Therefore, the chamber is adaptable to the protocol instead of having to adapt the protocol to the chamber. Pressure distribution/perfusion ring 22 fits within platform 24 and secured by a threaded cap having an indicator of rotational torque to prevent application of excessive force on the microaqueduct slide 18 and coverslip 32 that may cause breakage or other damage to the rigid components of flow chamber 10.

[0033] Microaqueduct slide 18 comprises an enlarged transparent member fabricated from an inert material such as glass or the like with peripheral geometry that is generally round with squared or flattened sides. A plurality of opposed shallow fluid perfusion grooves 20 are formed on the bottom surface of the slide member such as by etching, or the like. The fluid perfusion grooves 20 comprise generally T-shaped mirror image micro-channels which are spaced from one another and disposed such that the stems of the T-shaped micro-channels are axially aligned with one another, while the tops of the T-shaped micro-channels are disposed generally in a parallel relationship to one another. U.S. Pat. No. 4,974,952 includes a detailed description of an exemplary microaqueduct slide, and is hereby incorporated by reference. Grooved patterns may be customized to produce specific flow characteristics.

[0034] Referring next to FIG. 2, shown in phantom is a microscope 100 having a stage 103. On stage 103 is shown one embodiment of the novel specimen heating and retaining assembly 105 of the present invention discussed in more detail below. Located within the assembly 105 but not forming a part of the present invention, there is illustrated for informational purposes a specimen plane in which a specimen of interest for microscopic examination would be present. Also illustrated in FIG. 2 is a heating controller 109 which also does not form part of the present invention. While heating elements can vary widely and are not limiting to the present invention, for purposes of this discussion heating controller 109 is connected via electrically conductive wires 111 to a heating electroresistive heater to heat the specimen as discussed in more detail below.

[0035] Referring now to FIG. 3, there is illustrated in perspective cross section along the line II-II of FIG. 2, the novel heating and retaining assembly 5 of the present invention.

[0036] Here it is made clear the internal design of chamber 10. The point of contact with the mounting surface of stage adapter 36 is a peripheral specialty glass structure 25 on which platform 24 rest within the same plane as the specimen placed on coverslip 32. In this manner of specimen support, physical displacement in the “Z” axis plane due to thermal changes is minimized. It is also apparent that all components of the chamber numbers 12, 21, 26, 18, 28, 32, and 24 are “sandwiched” together to form a fluid optical cavity all resting on 25 within a stage adapter 36.

[0037] While the exemplary embodiments illustrated in the figures and described herein are presently preferred, it should be understood that these embodiments are offered by way of example only. Accordingly, the present application is not limited to a particular embodiment, but extends to various modifications that nevertheless fall within the scope of the appended claims. The order or sequence of any processes or method steps may be varied or re-sequenced according to alternative embodiments.

[0038] It is important to note that the construction and arrangement of the flow chamber as shown in the various exemplary embodiments is illustrative only. Although only a few embodiments have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter recited in the claims. For example, elements shown as integrally formed may be constructed of multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. Accordingly, all such modifications are intended to be included within the scope of the present application. The order or sequence of any process or method steps may be varied or re-sequenced according to alternative embodiments. In the claims, any means-plus-function clause is intended to cover the structures described herein as performing the recited function and not only structural equivalents but also equivalent structures. Other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments without departing from the scope of the present application.